The Construction Of GLS/IL-12 Recombinant BCG And Its Therapeutic Effects For Tumor Bearing Mice

Malignant tumor is one of the leading causes which endanger the people's health and lives in the world.Cancer has become the major cause of death among all kinds of diseases.Surgery,chemotherapy and radiation therapy are main methods of traditional treatments.With the development of molecular biology and biotechnology recently,gene therapy has become a promising therapeutic method to treat cancer besides traditional treatments.Gene therapy has become a focus of cancer treatments now.In this study,we constructed an eucaryotic co-expression plasmid pBM9 carrying human granulysin and murine IL-12 genes,transformed it into BCG,and recombinant BCG carrying GLS/IL-12 had been constructed; Animal model of murine melanoma was established and anti-tumor effect of recombinant BCG in murine melanoma model was observed by local intratumoral administration.Part 1 The construction of eucaryotic co-expression plasmid pBM9 carrying human granulysin leader peptide and murine IL-12 Objectives:To construct an eucaryotic co-expression plasmid pBM9 carrying human granulysin active peptide and murine IL-12 genes and determine its expression.Methods:The primer pairs including granulysin leader peptide DNA sequence were designed to amplify granulysin from the plasmid pZM03 carrying granulysin gene by polymerase chain reaction(PCR).PCR product was directed cloning into eucaryotic co-expression plasmid pBudCE4.1 to construct plasmid pBudCE4.1-S9K.pBudCE4.1-S9K plasmid was identified by DNA sequencing.Murine IL-12 gene from plasmid pZMO2 was subcloned into pBudCE4.1-S9K to construct eucaryotic co-expression plasmid pBudCE4.1-S9K/mIL-12.Mycobacteria replicon Orim from plasmid pZMO3 was subcloned into NheⅠsite of pBudCE4.1-S9K/mIL-12 to construct eucaryotic co-expression shuttle plasmid pBM9.pBM9 was tansfected into cells.In transfected cells and culture supernatant,the expressions of GLS and mIL-12 were detected by reverse transcriptase PCR(RT-PCR),immunocytochemistry and enzyme-linked immunosorbent assay(ELISA).Results:Granulysin gene fragment including leader peptide was obtained from pZM03 plasmid by PCR.The plasmid eucaryotic pBudCE4.1-S9K was identified by restrict endonuclease digestion and DNA sequencing respectively,and it was transfected into cells.The specific bands for 267 bp granulusin and 1632 bp mIL-12 were amplified from transfected cells by RT-PCR.The brown granules were detected in transfected cells by immunocytochemistry.Secretary products of mIL-12 were detected in culture supernatant by ELISA.Conclusions:Eukaryotic co-expression plasmid pBM9 carrying human GLS leader peptide and mIL-12 genes was constructed successfully.Part 2 Construction of the recombinant BCG carrying human granulysin and mIL-12 genesObjectives:To construct recombinant BCG carrying human granulysin and mIL-12 genes,and to identify the ability of submitting plasmids to eukaryotic cells.Methods:The pBM9 was transfected to BCG competence by electrotransformation to construct recombinant BCG.The plasmid of recombinant BCG was extracted for PCR identification;The plasmid stability of BCG was identified by culturing the recombinant BCG in Middlebrook 7H10 plate(Zeocin 40μg/ml).The recombinant BCG carrying pBMS,pBM12 was constructed in the same way seperately and was used as control bacteria in animal experiments.The ability of submitting plasmids to eukaryotic cells was determined by RT-PCR. Results:The plasmid pBM9 may be transduced into BCG with electrotransformation.The recombinant BCG can grow and passage in Middlebrook 7H10 plate(Zeocin 40μg/ml)and the specific bands for GLS and mIL-12 were detected by PCR.The recombinant BCG can be cultured at least 10 generations in low-salt LB plate without losing the plasmids,and then was transfected to mice macrophages.The specific bands for 267 bp granulusin and 1632 bp mIL-12 were amplified from transfected macrophages after 96 hours by RT-PCR.Conclusions:The recombinant BCG carrying pBM9,pBMS and pBM12 were constructed successfully.The recombinant BCG can be passaged at least 10 generations without losing the plasmids.The recombinant BCG can submit the genes carried in plasmids to mice macrophages.Part 3 Induction of immune response in mice by recombinant BCGObjectives:To observe immune response of normal mice induced by recombinant BCG.Methods:C57BL/6J inbred line mice were inoculated with 1×10~5 CFU/50μl recombinant BCG subcutaneously in tail.It was divided into 8 groups in the experiment,the mice were injected subcutaneously in tail with saline,BCG,pBM9 recombinant BCG,pBM12 recombinant BCG, pBMS recombinant BCG,pBM9 plasmid,pBM12 plasmid and pBMS plasmid respectively.The optimal time of inoculations was 0,7 and 10 day.Blood was obtained by removal of eyeballs,and then the mice were euthanized by decapitation.IL-12,IFN-γand TNF-αlevels were detected in serum according to the specification of ELISA kit.Differences of the cytokines levels were compared.Results:IFN-γlevel of serum in various recombinant BCG groups and various plasmid groups was significantly higher that in saline control group(P＜0.05).Moreover,IFN-γlevel of serum in pBM9 and pBM12 recombinant BCG groups was obviously higher that in BCG group (P＜0.05).IL-12 level of serum in various recombinant BCG groups and various plasmid groups was greater that in saline control group(P＜0.05). IL-12 level of serum in pBM12 recombinant BCG groups was remarkably greater than that in BCG group(P＜0.05).There was no difference about IL-12 level between the other two recombinant BCG groups and BCG group(P＞0.05).TNF-αlevel of serum in various recombinant BCG groups and various plasmid groups was noticeably higher than that in saline control group(P＜0.05).There was no difference about TNF-αlevel between various recombinant BCG groups and BCG group(P＞0.05).Conclusions:The recombinant BCG had the ability to induce cell immune responses of C57BL/6J. Part 4 Anti-tumor effect of eucaryotic co-expression recombinant BCG carrying human granulysin and murine IL-12 genesObjective:To establish animal model of murine melanoma and to observe anti-tumor effect of pBM9,pBM12 and pBMS recombinant BCG in murine melanoma model by local intratumoral administration.Methods:C57BL/6J inbred line mice were inoculated with 10~6 B16 cells(0.1 ml cells suspension)subcutaneously in right forelimb oxter,and the model was confirmed by pathological section and HE staining until the tumor reached 1 cm in size.The mice were treated with recombinant BCG by intratumoral administration.The optimal time to treat model mice was 1, 4,7 and 10 day after innoculation.The tumor-bearing mice were randomly divided into 5 groups,they were treated with saline,BCG,pBM9 recombinant BCG,pBM12 recombinant BCG,pBMS recombinant BCG by intratumoral administration respectively.At the next day after receiving B16 cell innoculation,each mouse was treated with 0.1ml corresponding liquid and it was repeated once every week for 4 weeks.Tumor-bearing mice were observed every two days,and tumor size was measured precisely in sliding caliper.Survival time was monitored.Pathological changes of tumor were observed in HE staining.The levels of IFN-γand IL-12 in serum were evaluated by ELISA. Results:The animal modal of murine melanoma could be established in C57BL/6J mice,and murine melanoma was observed by pathological detection.The optimal time for therapy was 1 day after tumor cells innoculation,and the effect was the best.The growth of tumor was suppressed after innoculation,and tumor-bearing mice with the treatment of pBM9 recombinant BCG(10~8/ml)showed smaller tumor size than that in saline control group(p＜0.05).A lot of lymphocyte infiltration near the tumor was found;The levels of IFN-γand IL-12 in serum in pBM9 recombinant BCG group were greater than that in saline control group(p＜0.05);Survival time was longer than that in saline control group(p＜0.05).Conclusion:The murine melanoma model was successfully established.The anti-tumor effect of pBM9 recombinant BCG in murine melanoma model was confirmed by local intratumoral administration.